Fig 2: Tfap2c, a direct target of Stat3, cooperates with MAPK pathway to induce Cdx2 expression.(A) qRT-PCR analysis of Cdx2 expression in B6-S3Y118F mESCs cultured in the indicated conditions. (B) Phase contrast images of B6-S3Y118F mESCs cultured in the indicated conditions for 5 days. (C) qRT-PCR analysis of Tead4 expression in scramble or Tead4 shRNAs-expressing B6-S3Y118F mESCs. (D) qRT-PCR expression of Cdx2 expression in scramble or Tead4 shRNAs-expressing B6-S3Y118F mESCs treated with GCSF for 24 hours. (E) qRT-PCR analysis of Gata3 expression in scramble or Tead4 shRNAs-expressing B6-S3Y118F mESCs treated with GCSF for 24 hours. (F) Phase contrast images of scramble (CN) or Tead4 shRNAs-expressing B6-S3Y118F mESCs cultured in the presence of GCSF for 7 days. (G) qPCR analysis of Tfap2c expression in B6-S3Y118F mESCs treated with GCSF or LIF for the indicated times. B6-S3Y118F mESCs were starved overnight in serum free medium prior to the treatments. (H) qPCR analysis of Tfap2c expression in B6-S3Y118F mESCs treated with GCSF for the indicated times. B6-S3Y118F mESCs were maintained in the LIF condition prior to GCSF treatment. (I) qPCR analysis of Tfap2c expression in B6-S3Y118F mESCs treated with GCSF or GCSF plus cycloheximide (CHX) for 1 hour. B6-S3Y118F mESCs were starved overnight in serum free medium prior to the treatment. (J) qRT-PCR analysis of Tfap2c expression in scramble or Tfap2c shRNAs-expressing B6-S3Y118F mESCs. (K) qRT-PCR analysis of Cdx2 expression in scramble or Tfap2c shRNAs-expressing B6-S3Y118F mESCs treated with GCSF for 24 hours. (L) Phase contrast images of scramble (CN) or Tfap2c shRNAs-expressing B6-S3Y118F mESCs cultured in the presence of GCSF for 7 days. (M) AP staining of scramble or Tfap2c shRNAs-expressing B6-S3Y118F mESCs cultured in the presence of GCSF for 7 days. (N) Model of mESC fate regulated by Stat3 activation level. Scale bars: 50 µm. Error bars represent the s.d. (n = 4).

Fig 3: Localisation of ESCs during chimaera formation. (A) Pie charts displaying percentage of SL or 2iL ESC deaths recorded at different locations in embryos. Top panels depict ESC localisation during day 1 of development prior to cavitation; bottom panels display day 2. Percentages are exclusive of deaths or divisions not clearly assigned to an embryonic location because of restricted visibility (20-30% of events). (B) Pie charts displaying percentages of SL or 2iL ESC divisions recorded at different locations in embryos, as for cell death events in A. (C-E) Representative images of ESCs localising to epiblast (C, 30/30), primitive endoderm (D, 2/30) or trophectoderm (E, 3/30) in late blastocyst chimaeras. Two left panels are maximum projections; remaining images are single planes. tdTomato-H2B, red; Sox17, blue; Cdx2, green. Scale bars: 30 µm in C-E.

Fig 4: Embryo invasion into PDCO or PD and trophoblast status on IVC day 2. a) Top view of the embryos invading into PDCO (n = 10) or PD (n = 10) on IVC day 2 by immunostaining using antibodies of CDX2 (red, labeling trophoblast) and Collagen I (green, labeling the top layer of PDCO). Scale bars, 100 µm. b) SEM images of the interfaces between embryos and the PDCO (n = 5) or PD (n = 5) substrates. The embryo- and TE-derived cells are colored green and dark blue, respectively. Scale bars, 50 µm (left panel) and 10 µm (right panel). c) Sectional SEM imaging the cross section of embryo invading into collagen gels of the PDCO. The detailed zooming information of collagen and embryo cells is indicated in the gray dotted box and red dotted box, respectively, which are also summarized by the Air force dotted line box schematic. White smoke, collagen; Iron: embryo cells. Scale bars, 50 µm (top panel) and 3 µm (bottom panel). d) Immunostaining of F-actin (green) for PDCO and PD embryos. Scale bars, 100 µm. e) Statistics of the embryonic spreading area of PDCO (n = 12) and PD (n = 12) embryos on IVC day 2, respectively. Two-tailed Student's t-test. ***P < 0.001. f) Immunostaining of embryos on PDCO (n = 12) and PD (n = 12) using antibodies of TFAP2C (gray, labeling TE) and DAPI (blue, labeling the nuclei). Scale bars, 100 µm. g) DIC images of embryos on PDCO or PD during AFM test. Scale bars, 50 µm. h) The statistic analyses of elastic modulus of the trophoblasts from embryos on PDCO or PD. n = 5 embryos for each condition. Two-tailed Student's t-test.***P < 0.001.

Fig 5: WW2 domain of YAP and PPQY motif of CDX2 are involved in YAP-CDX2 interaction. A Schematic illustration of the domain organization of YAP showing the various deletion constructs cloned and used for experimental purposes. The amino acid residue numbers are indicated at the top of each construct. CC, Coil–coil domain; TAD, transactivation domain; WW, tryptophan-rich domains. B Immunoprecipitation of CDX2 with either FLAG-tagged full-length YAP or various deletion constructs of YAP using lysates from TSCs transfected with either FLAG-tagged full-length YAP or various deletion constructs of YAP. Immunoprecipitation was done using anti-FLAG antibody followed by immunoblot with anti-CDX2 antibody. Immunoprecipitation using an isotype matched antibody was used as a negative control. IgG heavy chain was used as loading control. C Schematic illustration of the domain organization of CDX2 showing the region deleted for the experimental purpose. The amino acid residue numbers are indicated at the top of each construct. DBD, DNA-binding domain. D Either full-length CDX2 or ?CDX2 was transfected in SH-SY5Y cells along with full-length YAP and maintained for 48 h. Extracts were immunoprecipitated using anti-FLAG antibody and coimmunoprecipitation of YAP was analyzed using anti-YAP antibody. Total cell lysates were used as input samples in B and D